During studies on distribution of pneumolysin in various pneumococcal strains, the hemolytic activity was not detected in the cell lysate of type 8 strain maintained at CBER. To examine the pneumolysin activity of type 8 strains in the environment, several strains isolated from pneumonia patients were analyzed for hemolytic activity and by immunocytochemistry, using biotinylated enzyme and fluorescent antibody techniques. Among 8 strains of type 8, 7 had low or no hemolytic activity, while 1 strain showed high hemolytic activity. Similar results were also observed by immunocytochemistry; 7 strains had negative reactions, while 1 strain showed a positive response. The effects of several chemical agents including divalent ions, such as Ca++, Zn++, Mg++; chelating agents, EDTA, EDPA (ethylene dihydroxy phenyl acetic acid); and nucleotide phosphates, including ATP, ADP, cyclic AMP on the hemolytic activity of pneumolysin were examined. Divalent ions, EDTA and nucleotide phosphates showed no significant effects on the hemolytic activity. In contrast, EDPA, which is a chelating agent with specificity for ferric ions, inhibited the growth of pneumococci and pneumolysin activity.